PROJECT SUMMARY Cardiovascular disease is the leading cause of death in the United States. Millions of vascular bypass, angioplasty and stenting procedures are performed each year to repair or replace diseased blood vessels. Up to 15-50% of angioplasties, 16-30% of saphenous vein bypass grafts, and up to 90% of synthetic coronary bypass grafts fail due to intimal hyperplasia within 1-3 years. Intimal hyperplasia, triggered by endothelial injury and/or mechanical injury to the vessel wall induces platelet activation and stimulates secretion of SMC mitogens such as platelet-derived growth factor (PDGF). PDGF stimulates smooth muscle cell (SMC) proliferation, migration and extracellular matrix (ECM) synthesis; ultimately leading to vessel occlusion. Intimal hyperplasia can be prevented with medications that inhibit platelet aggregation and SMC proliferation, but there are currently no approved drugs that arrest or reverse intimal growth. A major obstacle to the development of new, more effective drugs is the lack of experimental models that mimic the characteristics of human intimal hyperplasia initiation and progression. Human vascular disease research predominantly depends on mouse models, although IH lesions in mice do not mimic human disease. As a result, drugs that succeeded in animal studies have failed in clinical trials. Cell culture and 3D tissues have been used as in vitro and ex vivo models to test vascular therapies. However, these systems are either in limited supply (e.g., human blood vessels), or fail to replicate the complex cell-cell, cell-ECM, and mechanical interactions characteristic of human blood vessels with intimal hyperplasia. To meet the need for vascular disease models for drug screening, our lab developed a novel, modular tissue fabrication approach that allows spatial customization of engineered tissue structure and function, giving us the unique ability to create 3D in vitro human vascular tissue that mimics the structural and mechanical features of human intimal hyperplasia. In the proposed project, we will fabricate tissue tubes from modular ring- shaped units of self-assembled human smooth muscle cells. We have also shown that gelatin microspheres can be incorporated within tissue rings during self-assembly to achieve growth factor delivery. To stimulate intimal hyperplasia in our vascular tissue model, PDGF-loaded microspheres will be incorporated within tissue rings to stimulate SMC proliferation. PDGF-loaded ring units will be fused with control SMC rings to form tubes. The tubes will be transferred to a custom bioreactor, seeded with endothelial cells and exposed to luminal fluid flow. The model will be validated by treating model human blood vessels with approved drugs to treat intimal hyperplasia. We will measure SMC proliferation and collagen synthesis, and the resulting effects on vascular tissue wall thickness, cross-sectional area and regional wall mechanics. We hypothesize that PDGF-loaded microspheres incorporated within engineered tissue rings and tubes will stimulate SMC proliferation and ECM synthesis in spatially defined regions that mimic the structure and function of human intimal hyperplasia.